Kibenge F S
Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Canada.
Am J Vet Res. 1992 Aug;53(8):1337-42.
Two nonoverlapping clones, pOH405 and pOH632, containing cDNA inserts in the VP2 coding region of genome segment A were selected from a cDNA library prepared from the double-stranded RNA genome of the OH strain of infectious bursal disease virus (IBDV) of serotype 2. Clone pOH405, which is located in the hypervariable segment of VP2, is 328 base pairs long, has nucleotide sequence homology of 72 to 73%, and amino acid sequence homology of 64 to 67% with IBDV strains of serotype 1. Clone pOH632, which is located in the highly conserved C-terminal part of VP2, is 230 base pairs long, has nucleotide sequence homology of 87 to 88%, and amino acid sequence homology of 100% with IBDV serotype 1. The lower detection limit of 32P-labeled probes prepared from both clones was 10 ng of OH-IBDV double-stranded RNA, using high-stringency conditions of hybridization (54 C, 50% formamide) and washing (55 C, 0.015M NaCl, 0.0015M trisodium citrate, pH 7.0, with 0.1% sodium dodecyl sulfate), and autoradiography for 24 hours. Under these conditions, the dot-blot hybridization assay for detection of serotype 2 IBDV double-stranded RNA, was 1,000 times more sensitive, using probe pOH632, but only 10 times more sensitive, using probe pOH405, compared with the assay for IBDV serotype 1, using the same probes. Thus, probe pOH632 could differentiate between the 2 IBDV serotypes by nucleic acid hybridization.
从2型传染性法氏囊病病毒（IBDV）OH株双链RNA基因组制备的cDNA文库中，挑选出两个不重叠的克隆体pOH405和pOH632，它们在基因组A节段的VP2编码区含有cDNA插入片段。位于VP2高变区的克隆体pOH405长328个碱基对，与1型IBDV毒株的核苷酸序列同源性为72%至73%，氨基酸序列同源性为64%至67%。位于VP2高度保守的C末端部分的克隆体pOH632长230个碱基对，与1型IBDV的核苷酸序列同源性为87%至88%，氨基酸序列同源性为100%。使用高严格度杂交条件（54℃，50%甲酰胺）和洗涤条件（55℃，0.015M氯化钠，0.0015M柠檬酸三钠，pH 7.0，含0.1%十二烷基硫酸钠）以及24小时放射自显影，由这两个克隆体制备的32P标记探针的最低检测限为10 ng的OH-IBDV双链RNA。在这些条件下，与使用相同探针检测1型IBDV的检测方法相比，使用探针pOH632检测2型IBDV双链RNA的斑点杂交检测方法灵敏度高1000倍，但使用探针pOH405时仅高10倍。因此，探针pOH632可通过核酸杂交区分这两种IBDV血清型。
